Gene transfer into primate embryonic stem cells using vsv-g pseudo type simian immunodeficiency virus vectors

ABSTRACT

Highly efficient gene transfer into primate-derived embryonic stem (ES) cells has successfully been achieved by using a simian immunodeficiency virus vector (SIV) pseudotyped with VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV) The present invention provides simian immunodeficiency virus vectors for gene transfer to primate ES cells. The method for gene transfer to primate ES cells using the vectors of the present invention is useful in, for example, research into embryology and disease, clinical applications, and experimental models for primates. The method is also useful in assaying and screening for genes and reagents able to enhance the specific differentiation of tissues or cells, and which are useful in preparing desired cells or tissues differentiated from ES cells.

TECHNICAL FIELD

[0001] The present invention relates to simian immunodeficiency virusvectors for gene transfer into primate embryonic stem cells.

BACKGROUND ART

[0002] Embryonic stem cells (hereinafter also referred to as “ES cells”)are undifferentiated cells having pluripotency and the ability toreplicate autonomously. Furthermore, it has been suggested that ES cellshave the ability to repair tissues after injury. Therefore, ES cells arebeing vigorously studied as being useful in screening therapeuticallyeffective substances for various diseases, and in the field ofregeneration medicine. Compared to murine ES cells, simian ES cells arecloser to those of humans, and therefore, they are expected to besuitable for use in human disease models.

[0003] Genetic engineering of ES cells will be extremely critical to thefuture application of ES cells in the treatment of various diseases andinjuries. To modify ES cell properties such as drug sensitivity, theability to proliferate and differentiate, and the like, stable genetransfer into the ES cell genome is often required. Retroviral vectorsthat integrate into the host genome are used routinely to achieve stablegene transfer. This is because, when cells such as ES cells that canproliferate and differentiate are used as targets, vectors will bediluted with each cell division if the introduced gene is not integratedinto the genome. However, when using the retroviral vector derived fromthe Moloney murine leukemia virus (MOMLV) , a commonly used genetransfer vector, the efficiency of gene transfer to murine ES cells islow (approximately a few percent), and the level of gene expressiondecreases with time. Recently, a retroviral vector derived from murinestem cell virus (MSCV) was used to improve the efficiency of genetransfer to murine ES cells (to 50% or higher). However, the problem ofreduced gene expression over time has not been solved (Cherry, S. R. etal. Mol. Cell Biol. 20:7419, 2000) Recently, it was shown that the useof the lentivirus vector, another vector which can integrate into thegenome, can further improve the efficiency of gene introduction intomurine ES cells (to 80% or higher) (Hamaguchi, I. et al. J. Virol.74:10778, 2000). However, in this report, expression of the introducedgene was only observed for a short time (a few days to about two weeks), and there was no record of long-term expression of the introducedgene.

[0004] Murine ES cells were used in all of the above-indicated reportsof gene transfer to ES cells. To date, there have been no reports ongene transfer to primate ES cells. However, an academic meeting reportindicated that gene transfer to primate ES cells is more difficult thangene transfer to murine ES cells. For example, the efficiency of genetransfer to primate ES cells has been reported to be approximately 1%using MOMLV vector, or approximately 5 to 10% using the MSCV vector(IMSUT Symposium for Stem Cell Biology, Tokyo, Japan 2000; Key StoneSympoia, Pluripotent Stem Cells: Biology and Applications, Durango,Colo., USA, 2001).

DISCLOSURE OF THE INVENTION

[0005] An objective of the present invention is to provide simianimmunodeficiency virus vectors for gene transfer to primate ES cells.Gene transfer to primate ES cells using the vectors of the presentinvention is useful for primate-related (including humans) research intoembryology and disease, clinical applications, experimental models, andsuch. This method is also useful for assaying and screening for genesand reagents which are required for specific differentiation of tissuesor cells and which are useful in preparing desired differentiatedtissues or cells from ES cells.

[0006] The present inventors developed a vector capable of gene transferinto primate ES cells. This vector was used in intensive studies toestablish a method for efficiently introducing foreign genes intoprimate ES cells. As a result, the inventors found that an SIV vectorpseudotyped with VSV-G protein, which is a surface glycoprotein ofvesicular stomatitis virus (VSV) , had the ability to transfer genesinto primate ES cells with a significantly high efficiency. Theefficiency of gene transfer into simian ES cells by theVSV-G-pseudotyped SIV vector was at least several to ten times greaterthan that into murine ES cells (FIG. 8). The efficiency of SIVvector-mediated transduction into ES cells increased depending on themultiplicity of infection (MOI). At a high MOI, genes were introducedinto almost all of the ES cells (FIG. 5). The introduced genes expressedstably over a long period in Cynomolgus monkey-derived ES cells intowhich an SIV vector containing a reporter gene ligated downstream of theCMV promoter was introduced. This expression hardly decreased even aftertwo months (FIG. 6).

[0007] Thus, the present inventors developed a pseudotyped SIV vectorwhich can transfer genes into primate ES cells and succeeded inestablishing a method of gene transfer into primate ES cells using thisvector. The present invention relates to simian immunodeficiency virusvectors for gene transfer to primate ES cells, and more specifically to:

[0008] (1) a recombinant simian immunodeficiency virus vectorpseudotyped with VSV-G and able to introduce a gene into a primateembryonic stem cell,

[0009] (2) the vector according to (1) , wherein the recombinant simianimmunodeficiency virus vector is derived from the agm strain,

[0010] (3) the vector according to (1) or (2) , wherein the recombinantsimian immunodeficiency virus vector is a self-inactivating vector,

[0011] (4) the vector according to any one of (1) to (3), wherein theprimate belongs to the Old World primates, of the familyCercopithecidae, genus Macaca,

[0012] (5) the vector according to any one of (1) to (4), which carriesa foreign gene in an expressible state

[0013] (6) the vector according to (5), wherein the foreign gene is agene encoding a protein selected from the group consisting of greenfluorescent protein, β-galactosidase, and luciferase,

[0014] (7) a method for introducing a gene into a primate embryonic stemcell, which comprises the step of contacting the cell with therecombinant simian immunodeficiency virus vector according to any one of(1) to (6),

[0015] (8) a primate embryonic stem cell into which the recombinantsimian immunodeficiency virus vector, according to any one of (1) to (6)has been introduced,

[0016] (9) a cell yielded by allowing the primate embryonic stem cellaccording to (8) to proliferate and/or differentiate, and

[0017] (10) a method for detecting the effect of an introduced gene onthe proliferation or differentiation of ES cells, which comprises thesteps of:

[0018] (a) introducing the vector, according to any one of (1) to (6)into a primate embryonic stem cell; and

[0019] (b) detecting the proliferation or differentiation of theembryonic stem cell.

[0020] As used herein, the term “viral vector” refers to a viralparticle capable of transferring nucleic acid molecules into a host. Theterm “simian immunodeficiency virus (SIV) vector” refers to a vectorhaving the SIV backbone. The term “having the SIV backbone” means thatthe nucleic acid molecules in the viral particle which constitutes thevector are based on the SIV genome. For example, one of the SIV vectorsof the present invention is a vector in which the nucleic acid moleculesin the virus particle comprise the packaging signal sequence derivedfrom the SIV genome. In the present invention, the simianimmunodeficiency virus (SIV) includes all SIV strains and subtypes.Isolated SIV strains include, but are not limited to, SIVagm, SIVcpz,SIVmac, SIVmnd, SIVsm, SIVsnm, and SIVsyk. The term “recombinant” viralvector refers to viral vectors constructed using recombinant genetechnology. Viral vectors which are constructed using DNA encoding theviral genome and packaging cells are included as recombinant viralvectors.

[0021] The term “VSV-G-pseudotyped vector” refers to vectors whichinclude the VSV-G protein, a surface glycoprotein of vesicularstomatitis virus (VSV). The VSV-G protein may be derived from anarbitrary VSV strain. For example, the VSV-G protein includes, but isnot limited to, proteins derived from the Indiana serotype strain (J.Virology 39: 519-528 (1981)). Alternatively, the VSV-G protein can be amodified VSV-G protein derived from the original protein by, forexample, substituting, deleting, and/or adding one or more amino acids.VSV-G-pseudotyped vectors can be prepared by allowing the VSV-G proteinto be present during viral production. Viral particles produced inpackaging cells can be pseudotyped with VSV-G by expressing VSV-G inthese cells. This can be facilitated by, for example, transfection of aVSV-G expression vector, or induced expression of the VSV-G geneintegrated into the host's chromosomal DNA. Since VSV-G protein ispresent on the membrane as a stable glycoprotein homotrimer, vectorparticles are hardly destroyed during purification and thus can beconcentrated to high concentrations using centrifugation (Yang, Y. etal., Hum Gene Ther: Sep, 6(9), 1203-13, 1995).

[0022] The pseudotyped retroviral vector of the present invention mayfurther contain envelope proteins from other viruses. For example, anenvelope protein derived from a virus which infects human cells ispreferred as such a protein. Such proteins include, but are not limitedto, retroviral amphotropic envelope proteins. For example, the envelopeprotein derived from murine leukemia virus (MuLV) 4070A strain can beused as such a retroviral amphotropic envelope protein. Alternatively,the envelope protein derived from MuMLV 10A1 can also be used (forexample, pCL-10A1 (Imgenex) (Naviaux, R. K. et al., J. Virol. 70:5701-5705 (1996)). Also included are proteins from the herpes virusfamily, such as the gB, gD, gH, and gp85 proteins derived from theherpes simplex virus, and the gp350 and gp220 proteins from the EBvirus. Proteins from the Hepadna virus family may include the S proteinof hepatitis B virus.

[0023] The simian immunodeficiency virus (SIV) was discovered as amonkey-derived HIV-like virus. Along with HIV, SIV forms the primatelentivirus group (E. Ido and M. Hayamizu, “Gene, Infection andPathogenicity of Simian Immunodeficiency Virus”, Protein, Nucleic acidand Enzyme, Vol. 39, No. 8, 1994). This group is further divided intofour subgroups: (1) The HIV-1 subgroup, containing HIV-1, the viruswhich causes human acquired immune deficiency syndrome (AIDS), andSIVcpz, which has been isolated from chimpanzees; (2) the HIV-2subgroup, containing SIVsmm isolated from Sooty Mangabeys (Cercocebusatys) , SIVmac isolated from rhesus monkeys (Macaca mulatta) , andHIV-2, which is less pathogenic in humans (Jaffar, S. et al., J. Acquir.Immune Defic. Syndr. Hum. Retrovirol., 16(5), 327-32, 1997); (3) theSIVagm subgroup, represented by SIVagm isolated from African greenmonkeys (Cercopithecus aethiops); and (4) the SIVmnd subgroup,represented by SIVmnd isolated from Mandrills (Papio sphinx).

[0024] There are no reports of SIVagm and SIVmnd pathogenicity innatural hosts (Ohta, Y. et al., Int. J. Cancer, 15, 41(1), 115-22, 1988;Miura, T. et al., J. Med. Primatol., 18(3-4), 255-9, 1989; M. Hayamizu,Nippon Rinsho, 47, 1, 1989). In particular, reports of infectionexperiments suggest that the TYO-1 strain of the SIVagm virus, which isused in the present Examples, is not pathogenic to crab-eating monkeys(Macaca facicularis) and rhesus monkeys (Macaca mulatta) , in additionto its natural hosts (Ali, M. et al, Gene Therapy, 1(6), 367-84, 1994;Honjo, Setal., J. Med. Primatol., 19(1), 9-20, 1990). There are noreports of SIVagm infection, pathogenesis or pathogenic activity inhumans. In general, primate lentiviruses have strictspecies-specificity, and there are few reports of cross-speciesinfection or pathogenesis from natural hosts. Where cross-speciesinfection does occur, the frequency of disease onset is normally low,and the disease progress is slow (Novembre, F. J. et al., J. Virol.,71(5), 4086-91, 1997). Accordingly, viral vectors based on SIVagm, andon the SIVagm TYO-1 strain in particular, are thought to be safer thanvectors based on HIV-1 or other lentiviruses, and are thus preferred foruse in the present invention.

[0025] The simian immunodeficiency virus vector of the present inventionmay contain a portion of a genomic RNA sequence derived from anotherretrovirus. Also included in the simian immunodeficiency virus vectorsof the present invention are vectors comprising a chimeric sequence,resulting from replacing a portion of the simian immunodeficiency virusgenome with, for example, a portion of the genomic sequence of anotherlentivirus, such as the human immunodeficiency virus (HIV), felineimmunodeficiency virus (FIV) (Poeschla, E. M. et al., Nature Medicine,4(3), 354-7, 1998) or caprine arthritis encephalitis virus (CAEV)(Mselli-Lakhal, L. et al., Arch. Virol., 143(4), 681-95, 1998).

[0026] In the retroviral vector of the present invention, the LTR (longterminal repeat) may also be modified. The LTR is a retrovirus-specificsequence, which is present at both ends of the viral genome. The 5′ LTRserves as a promoter, enhancing proviral mRNA transcription. Thus, itmay be possible to enhance mRNA transcription of the gene transfervector, improve packaging efficiency, and increase vector titer if theportion exhibiting th 5′ LTR promoter activity in the gene transfervector that encodes viral RNA genome packaged into viral particles, issubstituted with another promoter having stronger promoter activity.Furthermore, for example, in the case of lentiviruses, viral tat isknown to enhance 5′ LTR transcription activity, and therefore,substitution of the 5′ LTR for a promoter not present on the tat proteinwill enable the exclusion of tat from the packaging vector. The RNA ofviruses which have infected or invaded cells is reverse transcribed andthe subsequent, linking of the LTRs at both ends forms a closed circularstructure. Then, viral integrase couples with the linkage site and thisstructure is then integrated into cell chromosomes. The transcribedproviral mRNA is the region ranging from the 5′ LTR transcriptioninitiation site to the 3′ LTR polyA sequence located downstream. The 5′LTR promoter portion is not packaged in the virus particle. Thus, evenif the promoter is replaced with another sequence, the portionintegrated into target cell chromosomes is unchanged. Based on the factsdescribed above, substitution of the 5′ LTR promoter is thought toprovide a safer vector with a higher titer. Thus, substitution of thepromoter at the 5′ end of a gene transfer vector can increase the titerof a packagable vector.

[0027] Safety can be improved by preventing transcription of thefull-length vector mRNA in target cells. This is achieved using aself-inactivating vector (SIN vector) prepared by partially eliminatingthe 3′ LTR sequence. The lentivirus provirus invading the target cellchromosomes, has its 5′ end bound to the U3 portion of its 3∝ LTR. Thus,following reverse-transcription, transcripts of the gene transfer vectorare integrated into target cell chromosomes such that the U3 portion isat the 5′ end. From this point begins the transcription of RNA with astructure similar to the gene transfer vector transcripts. If there werelentivirus or related proteins in target cells, it is possible that thetranscribed RNA would be re-packaged and infect other cells. There isalso a possibility that the 3′ LTR promoter might express host geneslocated adjacent to the 3′ end of the viral genome (Rosenberg, N.,Jolicoeur, P., Retroviral Pathogenesis. Retroviruses. Cold Spring HarborLaboratory Press, 475-585, 1997). These are already considered to beproblems of retroviral vectors, and the SIN vector was developed as away of overcoming these problems (Yu, S. F. et al., Proc. Natl. Acad.Sci. USA, 83(10), 3194-8, 1986). When the 3′ LTR U3 portion is deletedfrom a gene transfer vector, target cells lack the promoters of 5′ LTRand 3′ LTR, preventing the transcription of the full-length viral RNAand host gene. Furthermore, since only the genes of interest aretranscribed from endogenous promoters, highly safe vectors capable ofhigh expression can be expected. Such vectors are preferable in thepresent invention. SIN vectors can be constructed according to methodsknown in the art, or methods as described in Examples 1 to 4.

[0028] One problem encountered in gene therapy using viral vectors thathave the LTR sequence in its genome, (including retroviral vectors) is agradual decrease in expression of the introduced gene. One factor behindthis may be that when such a vector is integrated into the host genome,a host mechanism methylates the LTR, suppressing expression of theintroduced gene (Challita, P. M. and Kohn, D. B., Proc. Natl. Acad. Sci.USA 91:2567, 1994). One advantage of SIN vectors is that LTR methylationhardly reduces gene expression level. This is because the vector losesmost of the LTR sequence upon integration into the host genome. Asdescribed in the Examples, an SIN vector, prepared by substitutinganother promoter sequence for the 3′ LTR U3 region of the gene transfervector, was revealed to maintain a stable expression for more than twomonths after gene transfer into primate ES cells. Thus, an SIN vectordesigned to self-inactivate by the modification of the LTR U3 region isespecially suitable in the present invention. Specifically, the presentinvention includes modified vectors in which one or more nucleotides inthe 3′ LTR U3 region have been substituted, deleted, and/or added. TheU3 region may simply be deleted, or another promoter may be insertedinto this region. Such promoters include, for example, the CMV promoter,the EF1 promoter, and the CAG promoter.

[0029] It is preferable to design the foreign gene encoded by the vectorof the present invention in such a way that it can be transcribed by apromoter other than LTR. For example, when the LTR U3 region is replacedwith a non-LTR promoter as described above, it is preferable that themodified LTR drives expression of the foreign gene. Alternatively, asshown in the Examples, the expression of a foreign gene can be inducedindependent of the LTR by placing a non-LTR promoter in a positiondifferent to the LTR region, and placing the foreign gene downstream ofthis position. The present invention showed that an SIV vector in whichthe expression of a foreign gene is regulated by a non-LTR promoterensures long-term stable expression of the foreign gene in ES cells.Thus, a vector in which a non-LTR promoter is placed upstream of aforeign gene, and where the foreign gene is transcribed under thecontrol of that promoter, is particularly suitable in the presentinvention. Such non-LTR promoters include the CMV promoter, EF1promoter, and CAG promoter. The CMV promoter in particular ispreferable. Such a vector is highly effective when constructed based onthe SIN vector described above.

[0030] A risk that has been pointed out concerning lentivirus vectorssuch as the HIV vector is that they may produce replicable viralparticles if the host genome already has the HIV provirus, andrecombination occurs between the foreign vector and the endogenousprovirus. This is predicted to become a serious problem in the future,when the HIV vector is used in HIV patients. The SIV vector used in thepresent invention has low sequence homology with HIV, and cannotreplicate as a virus because 80.6% of the virus-derived sequence hasbeen removed from the vector. Thus, this vector does hardly pose thisrisk and is safer than other lentivirus vectors. The preferred SIVvector of the present invention is a replication-incompetent virus fromwhich 40% or more, more preferably 50% or more, still more preferably60% or more, even more preferably 70% or more, and most preferably 80%or more of the genomic sequence of the original SIV has been removed.

[0031] Retroviruses can be produced by transcribing in host cells genetransfer vector DNA which contains a packaging signal. This allows theformation of virus particles in the presence of gag, pol and envelopeproteins. The packaging signal sequence encoded by the gene transfervector DNA should preferably be sufficient in length to maintain thestructure formed by the sequence. However, in order to suppress thefrequency of wild-type virus formation, which occurs due torecombination of the vector DNA packaging signal and the packagingvector supplying the gag and pol proteins, it is also necessary to keepsequence overlapping between these vector sequences to a minimum.Therefore, when it comes to the construction of the gene transfer vectorDNA, it is preferable to use a sequence which is as short as possibleand yet still contains the sequence essential for packaging, to ensurepackaging efficiency and safety.

[0032] For example, in the case of the SIVagm-derived packaging vectorused in the Example, the type of virus from which the signal to be usedis derived is limited to SIV, because HIV vectors are not packaged.However, the SIV-derived gene transfer vector is also packagable when anHIV-derived packaging vector is used. Thus, the frequency of recombinantvirus formation can be reduced if the vector particles are formed bycombining the gene transfer vector and packaging vector, where eachvector is derived from a different type of lentivirus. SIV vectors thusproduced are also included in vectors of this invention. In such cases,it is preferable to use combinations of primate lentiviruses (forexample, HIV and SIV).

[0033] In a preferred gene transfer vector DNA, the gag protein has beenmodified such that it is not expressed. Viral gag protein may bedetected by a living body as a foreign substance, and thus as apotential antigen. Alternatively, the protein may affect cellularfunctions. To prevent gag protein expression, nucleotides downstream ofthe gag start codon can be added or deleted, introducing modificationswhich will cause a frameshift. It is also preferable to delete portionsof the coding region of the gag protein. The 5′ portion of the codingregion of the gag protein is known to be essential for virus packaging.Thus, in a gene transfer vector, it is preferable that the coding regionfor the gag protein is deleted at the C terminus. It is preferable todelete as large a portion of the gag coding region as possible, so longas the deletion does not considerably affect the packaging efficiency.It is also preferable to replace the start codon (ATG) of the gagprotein with a codon other than ATG. The replacement codon can beselected appropriately so as not to greatly affect the packagingefficiency. A viral vector can be produced by introducing theconstructed gene transfer vector DNA, which comprises the packagingsignal, into appropriate packaging cells. The viral vector particlesproduced can be recovered from, for example, the culture supernatant ofpackaging cells.

[0034] There is no limitation on the type of packaging cell, as long asthe cell line is generally used in viral production. When used for humangene therapy, a human- or monkey-derived cell is suitable. Human celllines that can be used as packaging cells include, for example, 293cells, 293T cells, 293EBNA cells, SW480 cells, u87MG cells, HOS cells,C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, TE671cells, etc. Monkey cell lines include, for example, COS1 cells, COS7cells, CV-1 cells, BMT10 cells, etc.

[0035] The type of foreign gene to be inserted into the vector is notlimited. Such genes include nucleic acids which encode proteins, andthose which do not encode proteins, for example, antisense nucleic acidsor ribozymes. The gene may have a natural or an artificially designedsequence. Artificial proteins include the products of fusion with otherproteins, dominant-negative proteins (including soluble receptormolecules and membrane-bound dominant negative receptors), truncatedcell-adhesion molecules, and soluble cell-surface molecules. Inaddition, the foreign gene may be a marker gene to assess the efficiencyof gene transfer, stability of expression, and so on. Marker genesinclude genes that encode green fluorescent protein (hereinafter alsoreferred to as “GFP”), β-galactosidase, and luciferase. The GFP-encodinggene is particularly preferable.

[0036] The pseudotyped viral vectors of the present invention can besubstantially purified. The purification can be achieved using knownpurification and separation methods, such as filtration, centrifugation,and column purification. For example, a vector can be precipitated andconcentrated by filtering a vector solution with a 0.45-μm filter, andthen centrifuging it at 42500×g at 4° C. for 90 minutes.

[0037] If necessary, the pseudotyped retroviral vector of the presentinvention can be prepared as a composition by appropriately usingdesired pharmaceutically acceptable carriers or media in combination.The term “pharmaceutically acceptable carrier” refers to a material thatcan be administered in conjunction with the vector and does notsignificantly inhibit vector-mediated gene transfer. Specifically, thevector can be appropriately combined with, for example, sterilizedwater, physiological saline, culture medium, serum, and phosphatebuffered saline (PBS). The vector can also be combined with astabilizer, biocide, and such. A composition containing a pseudotypedretroviral vector of the present invention is useful as a reagent orpharmaceutical. For example, a composition of the present. invention canbe used as a reagent for gene transfer into ES cells, or as apharmaceutical for gene therapy.

[0038] Nucleic acids inserted into a vector of the present invention canbe introduced into the ES cells of primates, including humans, bycontacting the vector with the ES cells. The present invention relatesto a method for introducing a gene into primate ES cells, whichcomprises the step of contacting the cells with the vector of thepresent invention. The present invention also relates to the use of therecombinant simian immunodeficiency virus vector, pseudotyped withVSV-G, for gene transfer to primate ES cells. There are no particularlimitations as to the type of primate ES cell into which the gene isintroduced. For example, the desired simian ES cells can be used. Thereare about 200 types of monkeys known in the world. Higher primates arebroadly categorized into the following two groups:

[0039] (1) New World Primates

[0040] Marmosets (Cailithrix jacchus) are widely known, and used asexperimental primates. The development of New World primates and OldWorld Primates is essentially the same, although the structure of theirembryos and placentas does differ.

[0041] (2) Old World Primates

[0042] Old World primates are closely related to humans. Rhesus monkeys(Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are knownto belong to this group. Japanese monkeys (Macaca fuscata) belong to thesame genus (the genus Macaca) as cynomolgus monkeys. The development ofOld World primates is quite similar to that of humans.

[0043] As used herein, the term “monkey” or “simian” refers to primates,and specifically to New World and Old World primates. There are nolimitations as to the type of simian ES cell into which genes areintroduced using a vector of the present invention. Such simian ES cellsinclude marmoset ES cells (Thomson, J. A. et al., Biol. Reprod. 55,254-259, (1996)), rhesus monkey ES cells (Thomson, J. A. et al., Proc.Natl. Acad. Sci. U.S.A. 92, 7844-7848, (1995)), and cynomolgus monkey EScells (see Examples). Since Old World primates are closely related tohumans and their development is similar to that of humans, they can beused as models to reflect human diseases, and as screening systems fortherapeutics for various diseases. Thus, it is preferable to use simianES cells derived from Old World primates, especially monkeys belongingto the genus Macaca, such as the Japanese monkey, the rhesus monkey, andthe cynomolgus monkey for introduction of a vector of the presentinvention.

[0044] Primate ES cells can be prepared by a known method or accordingto the method described herein in the Examples. For example, ES cellscan be obtained from developing blastocysts (for example, see thepamphlet WO 96/22362). Specifically, ES cells can be established, forexample, by culturing blastocyst-derived inner cell masses on feedercells or with the leukemia inhibitory factor [LIF, also referred to as“differentiation inhibiting factor (DIF)”].

[0045] Such feeder cells include primary cultures of fetal fibroblastsfrom mice after a gestation of 12 to 16 days, cells obtained by treatingmouse fetal fibroblast cell lines, such as STO cells, with mitomycin C,X-rays, or the like. Mouse-derived feeder cells can be prepared on alarge scale and are thus advantageous in experiments and such. Thefeeder cells can be prepared, for example, according to the methoddescribed below in the Examples. The feeder cells are plated, forexample, on gelatin-coated culture containers using MEM (MinimumEssential Medium Eagle). The feeder cells can be plated in culturecontainers such that they are fully confluent. The MEM medium in theculture container is changed to ES cell culture medium (see Table 3 inExample 6) and the inner cell masses are plated onto the feeder cells.

[0046] Genes can be introduced into primate ES cells using a vector ofthe present invention by using a method that comprises the step ofcontacting the vector with primate ES cells. Specifically, for example,ES cells into which a gene is to be introduced are plated on culturedishes covered with feeder cells, and then an SIV vector is added. Theefficiency of gene transfer can be improved by simultaneously addingpolybrene, for example, at a concentration of about 8 μg/ml. Genetransfer can be carried out at an MOI (multiplicity of infection: thenumber of infectious viral particles per cell) of, for example, 0.1 to1000, more preferably 1 to 100, yet more preferably 2 to 50 (forexample, 5 to 10). Normally, genes can be introduced into most ES cellsby a single addition of the vector, without the need for repeatadditions. The vector of the present invention has the advantage that itcan achieve exceedingly high gene transfer efficiency withoutRetroNectin™.

[0047] In addition, the present invention relates to primate ES cells inwhich the VSV-G-pseudotyped viral vector of the present invention hasbeen introduced, and the cells yielded through proliferation and/ordifferentiation of these ES cells. The differentiation of ES cells canbe induced by, for example, adding known differentiation/growth factorssuch as cytokines, or substrates such as extracellular matrices, byco-culturing with other cells, or by transplanting the cells intoindividuals, (Hitoshi Niwa “ES cell differentiation fate decisionmechanism” Protein, Nucleic acid and Enzyme 45: 2047-2055, 2000;Rathjen, P. D. et al., Reprod. Fertil. Dev. 10: 31-47, 1998).

[0048] For example, methods for inducing the differentiation of cellsderived from extraembryonic tissue include the following: Extraembryonicendoderm: Formation of embryoid bodies Trophectoderm: Suppression ofOct-3/4 expression

[0049] Methods for inducing differentiation of cell types derived fromundifferentiated cells include the following: Primitive ectoderm:Formation of embryoid bodies Culture supernatant of HepG2

[0050] Methods for inducing differentiation of cells derived from theectoderm include the following: Neurons: Formation of embryoid bodies +treatment with retinoic acid Formation of embryoid bodies + bFGFFormation of embryoid bodies + treatment with retinoic acid + aselection of Sox2-positive cells Glial cells: Formation of embryoidbodies + treatment with retinoic acid Formation of embryoid bodies +bFGF Epithelium cells: Formation of embryoid bodies

[0051] Methods for inducing differentiation of cells derived from neuralcrest cells or such include the following: Pigment cells: Formation ofembryoid bodies OP9 + ST2 + dexamethasone + SOL Steroid-producing cells:Over-expression of SF1

[0052] Methods for inducing differentiation of cells derived from themesoderm include the following: Hematocytes (Hematopoietic Formation ofembryoid bodies + IL-3 + stem cells): IL-6 + feeder cells OP9 + feedercells A selection of flk1-positive cells Vascular endothelial cells: Aselection of flk1-positive cells Osteoclasts: Formation of embryoidbodies + retinoic acid treatment Cardiac muscle cells: Formation ofembryoid bodies Formation of embryoid bodies + a selection ofαMHC-positive cells Skeletal muscle cells: Formation of embryoid bodiesSmooth muscle cells: Formation of embryoid bodies Formation of embryoidbodies + DMSO Adipocytes: Formation of embryoid bodies + treatment withretinoic acid + insulin + T3

[0053] Methods for inducing differentiation of cells derived fromendoderm include the following: Insulin-producing cells: Formation ofembryoid bodies

[0054] ES cells in which a gene has been introduced using a pseudotypedviral vector of the present invention, and cells, tissues, organs, andsuch, that have been differentiated from these ES cells, are useful inassays of and in screening for various pharmaceutical agents. Forexample, gene transfer to primate ES cells can be used to screen for andassess the efficacy of pharmaceutical agents, genes involved in thespecific differentiation of tissues or cells, preferably primate-derivedtissues or cells, and the like. The present invention provides a methodfor screening for genes or pharmaceutical agents involved in thespecific differentiation of tissues or cells. The present inventionprovides a method for detecting the effect of an introduced gene on theproliferation or differentiation of ES cells, which comprises the stepsof: (a) introducing a vector of the present invention into primate EScells, and (b) detecting the proliferation or differentiation of theseES cells. The vector can be introduced into ES cells by contactingprimate ES cells of interest with a recombinant simian immunodeficiencyvirus vector of the present invention. The proliferation of ES cells canbe tested by a known method including counting the number of cells, ormeasuring mitochondrial activity using, for example, an MTT assay. Thedifferentiation of ES cells can be detected by testing the expression ofknown differentiation marker genes, morphological or biochemical assaysof cells or tissues, or the like (Satoshi Niwa “ES cell differentiationfate decision mechanism” Protein, Nucleic acid and Enzyme 45: 2047-2055,2000; Rathjen, P. D. et al., Reprod. Fertil. Dev. 10: 31-47, 1998). Thegene transfer vector can contain desired foreign genes whose effects areto be determined. The vector can be used without a foreign gene whendetermining the effect of introducing the vector itself, for example,when used as a negative control. Genes influencing the proliferation ordifferentiation of primate ES cells can be assessed or selected byscreening using the detection method described above. Such screeningscan be achieved by a method which comprises: the same steps (a) and (b)as in the above description of the detection method, followed by step(c) where an introduced gene with the activity of regulating theproliferation or differentiation of ES cells is selected. Such screeningmethods are also included in the above-described method for detectingthe effect of the gene transfer of the present invention.

[0055] One example of screening using such a method is screening forgenes which differente primate ES cells into functional cells.

[0056] When, for example, determining which of genes A, B, C, D, and Eis essential for the differentiation of pancreatic β cells from primateES cells, in what combination these genes are most effective, or in whatorder they are preferably introduced, a method that simply andefficiently transfers genes into primate ES cells is useful. The vectorsof the present invention meet such requirements. For example, after thevectors of the present invention are constructed for the expression ofthe genes A, B, C, D, and E, the genes are introduced in variouscombinations or orders into primate ES cells or into cellsdifferentiated from these ES cells. The effect of gene transfer can beassessed by detecting the differentiation of the cells into which thegenes have been introduced.

[0057] In addition, the vectors of the present invention are useful, forexample, in predicting the presence of side effects in gene therapy thatcomprises introducing a particular gene into a body.

[0058] The toxicities and side effects of gene X on each organ or tissuecan be roughly estimated by carrying out experiments that compriseintroducing gene X into mice or monkeys. However, it is difficult todetermine the influence of gene X on the stem cells of each tissue usingconventional methods. There is a possibility that gene X may inhibit thedifferentiation into functional cells of a particular tissue's stemcells. For example, if the gene inhibits the differentiation of hepaticstem cells, then its inhibitory effect is revealed for the first timewhen a person is affected with hepatitis or undergoes hepatectomy. Thus,hepatic stem cell differentiation is inhibited by gene X, and thedesired hepatic regeneration does not proceed; a serious problem. Suchproblems can not necessarily be predicted from the results ofconventional animal experiments. Using a vector of the presentinvention, gene X can be introduced into primate ES cells withexceedingly high efficiency. The safety of gene X at various stages ofdifferentiation can be assessed by differentiating the gene-introducedES cells into various tissue stem cells and further into functionalcells.

[0059] Assays and screenings using vectors of the present invention areuseful in, for example, research into embryology and disease, clinicalapplications, and experimental models involving primates, and humans andmonkeys in particular. The vector of the present invention also enablesscreening for genes and reagents useful in preparing desireddifferentiated cells or tissues.

[0060] In the screening method described above, the specificdifferentiation of ES cells into desired tissues or cells can beassessed by, for example, using the expression of a marker specific tothe desired tissue or cell type as an index. This marker includestissue- or cell-specific antigens. For example, markers for neuralprogenitor cells include nestin, which is an intermediate filament. Themarker can be detected by applying an antibody against that specificmarker in conventional ELISA, immuno-staining, or such. The marker canalso be detected by applying a nucleic acid encoding the marker inconventional RT-PCR, DNA array hybridization, or such. The term “nucleicacid” refers to genomic DNA, RNA, mRNA, cDNA, and such. Genes andreagents obtained by the screening method are included in the presentinvention.

[0061] Furthermore, ES cells in which a pseudotyped retroviral vector ofthe present invention has been introduced, and cells and tissuesdifferentiated from those ES cells, are also included in the presentinvention. Such differentiated cells and differentiated tissues can beidentified by examining the expression of the above-mentioned markerspecific to a tissue or cell type, or through morphological observationsof the tissues or cells.

[0062] The viral vector of the present invention can be used in genetherapy for any primate genetic disease. There is no limitation as tothe type of disease to be treated. For example, diseases to be treatedand their single causative genes include: Gaucher disease(β-cerebrosidase (chromosome 20)); hemophilia (blood coagulation factorVIII (X chromosome) and blood coagulation factor IX (X chromosome)) ;adenosine deaminase deficiency (adenosine deaminase); phenylketonuria(phenylalanine hydroxylase (chromosome 12)); Duchenne muscular dystrophy(dystrophin (X chromosome)); familial hypercholesterolemia (LDL receptor(chromosome 19)); and cystic fibrosis (chromosomal translocation of theCFTR gene). Target diseases in which multiple genes are thought to beinvolved include neurodegenerative diseases such as Alzheimer's diseaseand Parkinson's disease, ischemic encephalopathy, dementia, andintractable infections such as AIDS.

[0063] In addition, cells, tissues and organs differentiated fromgene-introduced ES cells can be used in treating diseases. For example,a disease caused by a gene deficiency or lack can be treated bycompensating for the deficient gene by integrating that gene into thechromosome of primate ES cells, and transplanting the cells into a body,thus making up for a shortage of an enzyme, growth factor, or such inthe circulating blood. Gene therapy associated with organtransplantation can be undertaken by replacing the histocompatibilityantigen of the non-human animal donor with that of a human. In this way,the success rate of xenografts can be improved.

[0064] When the ES cells into which a gene has been introduced using avector of this invention are monkey-derived, the ES cells can betransplanted into disease model monkeys, providing useful models forhuman disease treatment. Many disease model monkeys are known forvarious human diseases. For example, model monkeys for human Parkinson'sdisease can be produced artificially; many naturally diabetic monkeysare bred as accurate models of human diabetes; and SIV infection inmonkeys is well known to serve as an accurate model of HIV infection inhumans. For such diseases, a system where simian ES cells aretransplanted to disease model monkeys as a preclinical test, prior tothe clinical application of human ES cells, is exceedingly useful.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1 is a diagram showing an outline of a lentivirus vectorsystem which uses the monkey immunodeficiency virus clone SIVagmTYO1.

[0066]FIG. 2 is a diagram showing the structure of the SIVagm genetransfer vector in which the U3 region, a promoter sequence of 5′ LTR,has been substituted with another promoter sequence.

[0067]FIG. 3 is a diagram showing the structure of the SIVagm genetransfer vector, in which the U3 region of 3′ LTR has been substitutedwith another promoter sequence. It also shows the structure of the U3promoter region of the 5′ LTR expected to be produced as a result ofreverse transcription of the vector in target cells.

[0068]FIG. 4 shows a diagram of the structure of the SIN vector whichcontains EGFP as a reporter (pGCL3C/CMVL.U3G2/RREc/s/CMVEGFP/3′ LTR,,U3)(also abbreviated to “SIN-GFP/SIV” or “SIN CMV EGFP”). The 3′ U3 wasremoved to obtain the self-inactivating vector (SIV vector).

[0069]FIG. 5 is a diagram showing the time course of EGFP expression inCMK-1 strain ES cells in which the EGFP gene had been introduced usingthe SIV vector. The horizontal axis indicates the number of days aftergene transfer (day 0). The vertical axis indicates the percentage ofEGFP-expressing CMK-1 cells. Values for the efficiency of CMK-1 genetransfer have been corrected for the contribution of feeder cellcontamination, using the method indicated in the Examples:“normalization equation for the efficiency of gene transfer for CMK-1”.The efficiency of gene transfer was dependent on MOI and was exceedinglyhigh two days after gene transfer; at MOI=100 the efficiency was 90% orhigher; at MOI=10 it was about 80%; and at MOI=1 it was about 60%. Thishigh transgenic efficiency lasted for at least about two months.

[0070]FIG. 6 is a diagram showing the time course of mean EGFPfluorescence intensity in CMK-1 strain ES cells into which the EGFP genehad been introduced via the SIV vector. The horizontal axis indicatesthe number of days after gene transfer (day 0). The vertical axisindicates mean EGFP fluorescence intensity (--) obtained by FACSanalysis. The mean fluorescence intensity of EGFP-expressing cells washardly reduced over about two months..

[0071]FIG. 7 shows micrographs of fluorescent images of EGFP expressionin CMK-1 strain ES cells into which a gene had been introduced using theSIV vector.

[0072] Top panel: Observation under a fluorescent microscope of ES cells21 days (day 21) after gene transfer. CMK-1 cells emitting EGFPfluorescence stand out as islets.

[0073] Bottom panel: Observation under a fluorescent microscope of EScells 62 days (day 62) after gene transfer. CMK-1 cells still emittingEGFP fluorescence stand out as islets.

[0074]FIG. 8 is a diagram indicating the efficiencies of SIVvector-mediated gene transfer into murine ES cells and simian ES cells(CMK-1 strain). The vertical axis indicates the efficiency (%) of genetransfer into ES cells, normalized for the contribution of feeder cellcontamination. Gene transfer into simian ES cells was more efficientthan that into murine ES cells. When an SIV-based vector is used, it ispredicted that cells from primates, such as SIV's natural host monkeys,will allow for more efficient gene transfer than cells from otherspecies, such as murine cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0075] The present invention is illustrated in detail below withreference to Examples, but it should not be construed as being limitedthereto. All of the literature cited throughout this specification isincorporated herein by reference.

[EXAMPLE 1] Generation of SIV Vectors

[0076] SIVagmTYO1 comprising a clone of an African green monkey-derivednonpathogenic immunodeficiency virus was used in the generation of avector system. FIG. 1 shows the outline of the vector system. Allnucleotide numbers are indicated below, with the transcriptioninitiation site of the viral RNA as +1. pSA212 in which SIVagmTYO1 hadbeen inserted was used as a plasmid (J. Viol., vol. 64, pp307-312,1990). All ligation reactions were carried out using Ligation High(Toyobo) according to the attached instructions.

[0077] a. Generation of a Packaging Vector

[0078] First, a DNA fragment corresponding to the region (5337-5770)containing vif and the first exon of tat/rev was obtained by PCR, usingpSA212 as a template, and using primers 1F (SEQ ID NO: 1) and 1R (SEQ IDNO: 2). A DNA fragment with an EcoRI site at its 3′ end was prepared bydesigning a PCR primer with an EcoRI restriction enzyme site. Afterdigestion with BglII and EcoRI, the PCR fragments were purified usingagarose gel electrophoresis and the Wizard PCR Preps DNA PurificationSystem (Promega). The DNA fragments resulting from the above procedure,together with a DNA fragment encoding the gag/pol region (including theregion from the XhoI site (356) to the BglII site (5338)), were ligatedat the XhoI-EcoRI site of pBluescript KS+ (Stratagene). Then, PCRamplification was performed for a DNA fragment corresponding to theregion containing the Rev responsive element (RRE) and the second exon(6964-7993) of tat/rev. In a similar manner as for the PCR fragmentdescribed above, PCR was carried out using pSA212 as a template andusing primers 2F (SEQ ID NO: 3) and 2R (SEQ ID NO: 4) to add a NotI siteat the 3′ end. After digestion with EcoRI and NotI, the DNA fragment waspurified and inserted at the EcoRI-NotI site of pBluescript KS+ intowhich gag-tat/rev had been inserted.

[0079] DNA fragments containing a splicing donor (SD) site weresynthesized (sequence 3F (SEQ ID NO: 5) and 3R (SEQ ID NO: 6)). At thetime of synthesis, an XhoI site and an SalI site were integrated intothe DNA at the 5′ and 3′ ends respectively, and then the DNA wasinserted at the XhoI site of the above-mentioned pBluescript KS+, intowhich gag-RRE-tat/rev had been inserted. The resulting plasmid wasdigested with XhoI and NotI, and the fragment containing the region fromSD to tat/rev was purified. The fragment was then inserted at theXhoI-NotI site of a plasmid into which an XhoI/NotI linker (sequence 4F(SEQ ID NO: 7) and 4R (SEQ ID NO: 8)) had been inserted at the EcoRIsite of pCAGGS (Gene, vol. 108, pp193-200, 1991). The plasmid obtainedvia the above method was used as a packaging vector (pCAGGS/SIVagmgag-tat/rev).

[0080] b. Generation of Gene Transfer Vectors

[0081] PCR was conducted using pSA212 as a template, and using thefollowing primers: primers 5-1F (SEQ ID NO: 9) and 5-1R (SEQ ID NO: 10)to amplify the SIVagmTYO1-derived 5′ LTR region (8547-9053+1-982,including a KpnI site at the 5′ end, and an EcoRI site at the 3′ end);primers 5-2F (SEQ ID NO: 11) and 5-2R (SEQ ID NO: 12) to amplify the RRE(7380-7993, including an EcoRI site at the 5′ end, and a SacII site atthe 3′ end) ; and primers 5-3F (SEQ ID NO: 13) and 5-3R (SEQ ID NO: 14)to amplify the 3′LTR (8521-9170, including NotI and BamHI sites at the5′ end, and a SacI site at the 3′ end). Furthermore, PCR was conductedusing pEGFPC2 as a template, and using primers 6F (SEQ ID NO: 15) and 6R(SEQ ID NO: 16) to amplify pEGFPC2-derived (Clontech) CMV promoter andthe region encoding enhanced green fluorescent protein (hereinafter alsoreferred to as EGFP) (1-1330; including a SacII site at the 5′ end, anda translational stop codon, a NotI site and a BamHI site at the 3′ end).The four types of PCR fragments respectively were digested with pairs ofrestriction enzymes: KpnI and EcoRI, EcoRI and SacII, BamHI and SacI,and SacII and BamHI, and then purified. They were then ligated betweenthe KpnI-SacI site of pBluescript KS+in the following order:5′LTR→3′→LTR→RRE and CMV promoter. EGFP(pBS/5′LTR.U3G2/RREc/s/CMVFEGFP/WT3′LTR). In order to insert theβ-galactosidase gene used as a reporter gene, DNA fragments containingthe 5′LTR region and 3′ LTR region respectively were prepared using PCRas described above. After restriction enzyme digestion with both KpnIand EcoRI, and both NotI and SacI respectively, the DNA fragments werepurified, and then inserted into the pBluescript KS+ at the KpnI-EcoRIsite and the NotI-SacI site respectively (pBS/5′LTR.U3G2/WT3′LTR). ANotI fragment containing the region encoding pCMV-beta β-galactosidase(Clontech) (820-4294) was inserted into the plasmid at the NotI site(pBS/5′ LTR.U3G2/beta-gal/WT3′ LTR). Then, an RRE sequence (6964-8177;including an EcoRI site at the 5′ end and a NotI site at the 3′ end),which had been amplified by PCR using pSA212 as a template and usingprimers 7-1F (SEQ ID NO: 17) and 7-1R (SEQ ID NO: 18) , was inserted atthe EcoRI-NotI site of plasmid pBS/5′ LTR.U3G2/beta-gal/WT3′ LTR (pBS/5′LTR.U3G2/RRE6/tr/beta-gal/WT3′ LTR). The RRE sequence was cut out withEcoRI and NheI prior to the insertion of the RRE sequence (7380-7993;including an EcoRI site at the 5′ end and a NheI site at the 3′ end)which had been amplified by PCR using pSA212 as a template and usingprimers 7-2F (SEQ ID NO: 19) and 7-2R (SEQ ID NO: 20). After theresulting plasmid (pBS/5′ LTR.U3G2/RREc/s/beta-gal/WT3′ LTR) wasdigested with NheI and SmaI and blunt ended, a CMV promoter region(8-592; blunt ended AseI-NheI fragment) derived from pEGFPN2 (Clontech)was inserted therein (pBS/5′ LTR.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR). Allblunting reactions were performed using a Blunting High (Toyobo)according to the attached instructions. The plasmids pBS/5′LTR.U3G2/RREc/s/CMVFEGFP/WT3′ LTR and pBS/5′LTR.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR were digested with KpnI and SacIrespectively to provide DNA fragments containing the region between the5′ LTR and the 3′ LTR. The fragments were inserted into the pGL3 Controlvector (Promega) at the KpnI-SacI site for use as a gene transfer vector(pGL3C/5′LTR.U3G2/RREc/s/CMVFbeta-gal/WT3′LTR orpGL3C/5′LTR.U3G2/RREc/s/CMVFEGFP/WT3′LTR).

[EXAMPLE 2] Modification of 5′LTR

[0082] The transcriptional activity of 5′LTR from lentivirus generallydepends on the presence of Tat protein, which is a virus-derived factor.Thus, to eliminate Tat dependence as well as to enhance vector titer byreplacement with a promoter sequence with stronger transcriptionalactivity, an SIVagm gene transfer vector was generated. In this SIVagmgene transfer vector, the U3 region, a promoter sequence of the 5′LTR,was replaced with another promoter sequence (FIG. 2).

[0083] The replacement of the 5′LTR with a chimeric promoter wasachieved as follows. A fragment containing the region downstream of theTATA box on the 5′LTR through to the gag region (9039-9170+1-982) wasamplified by PCR using pSA212 as a template and using a series ofprimers 9-1F to 3F (SEQ ID NOS: 21-23) and primer 9R (SEQ ID NO: 24).Further, fragments containing the CMVL promoter (derived from pCI(Promega); 1-721) were amplified by PCR using pCI as a template and apair of primers 10-1F (SEQ ID NO: 25) and 10-1R (SEQ ID NO: 26).Fragments containing the CMV promoter (derived from pEGFPN2 (Clontech);1-568) were amplified by PCR using pEGFPN2 as a template and a pair ofprimers 10-2F (SEQ ID NO: 27) and 10-2R (SEQ ID NO: 28). Fragmentscontaining the EF1α promoter (nucleotides 2240-2740 from pEF-BOS(Nucleic Acids Research, vol. 18, p5322, 1990)) were amplified by PCRusing pEF-BOS as a template and a pair of primers 10-3F(SEQ ID NO: 29)and 10-3R(SEQ ID NO: 30). Fragments containing the CA promoter(nucleotides 5-650 from pCAGGS) were amplified by PCR using pCAGGS as atemplate and a pair of primers 10-4F(SEQ ID NO: 31) and 10-4R (SEQ IDNO: 32). After amplification, fragments containing the 5 ′LTR were mixedwith each of the above fragments which each contained a promoter. Theprimer (10-1F(SEQ ID NO: 25), 10-2F (SEQ ID NO: 27), 10-3F (SEQ ID NO:29), or 10-4F (SEQ ID NO: 31)) corresponding to the 5′ end of eachpromoter, and the primer corresponding to the 3′ end of the 5′LTR (9R)were added thereto. PCR was then performed for another ten cycles toobtain DNA fragments of a chimeric promoter which consisted of each ofthe four types of promoters and the 5′LTR. The resulting DNA fragmentswere inserted into a gene transfer vector (PGL3C/5′LTR.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR) at the KpnI-EcoRI site(pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR,pGL3C/CMV.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR, pGL3C/EF1α.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR,pGL3C/CAG.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR).

[EXAMPLE 3] Modification of the 3′ LTR

[0084] A self-inactivating (SIN) vector was constructed by removing aportion from the 3′ LTR sequence such that transcription of full-lengthvector mRNA in target cells was prevented, and safety was improved. Inlentivirus vectors it has been demonstrated that the U3 region, whichserves as a promoter sequence in the 3′ LTR, can be integrated into the5′ LTR U3 promoter region when reverse-transcribed in target cells.Therefore the 3′ LTR U3 region of the gene transfer vector plasmid canserve as the 5′ LTR U3 promoter involved in gene expression in thegenome of target cells (FIG. 3). Thus, a vector was prepared in whichthe 3′ LTR U3 region of the SIVagm gene transfer vector was replacedwith another promoter sequence (FIG. 3). In addition, in order to testwhether the 5′ LTR promoter sequence can be deleted in target cells, avector in which the 3′ LTR U3 region of the SIVagm gene transfer vectorhad been deleted was also prepared.

[0085] The modification and deletion of the U3 promoter sequence of the3′LTR was achieved as follows. A DNA fragment without the 3′LTR U3 wasamplified by PCR using pSA212 as a template and using primers 11F (SEQID NO: 33) and 11R (SEQ ID NO: 34). Further, 3′LTRs, in which the U3region had been replaced with another promoter, were amplified by PCRusing a series of primers 12-1F to 3F (SEQ ID NOS: 35-37) and primer 12R(SEQ ID NO: 38), as well as using as templates, each of vector plasmids,obtained by the method described in Example 2, into which a chimericpromoter had been inserted:

[0086] pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR, pGL3C/EF1α.

[0087] U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR, and

[0088] pGL3C/CAG.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR. The resulting DNAfragments provided by PCR were digested with SalI and SacI, purified,and inserted into pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/WT3′ LTR at theSalI-SacI site

[0089] (pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/3′LTRdeltaU3,

[0090] pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/CMVL.R,

[0091] pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/EF1α. R, and

[0092] pGL3C/CMVL.U3G2/RREc/s/CMVFbeta-gal/CAG.R) respectively.

[0093] In addition, an SIN vector containing EGFP as a reporter(pGCL3C/CMVL.U3G2/RREc/s/CMVEGFP/3′LTR,,U3) (FIG. 4) was constructedfrom EcoR1-BamHI-treated pGCL3C/CMVL.U3G2/RREc/s/CMVF β-gal/3′LTR,,U3 byreplacing a fragment containing β-gal with the EcoRI-BamHI fragment ofthe product obtained by PCR using pEGFP-C2 (Clontech) as a template, andthe primers EGFPFG2Eco (ATCGGAATTCGGCCGCCATGGTGAGCAAGGGCGAGGAGCT/SEQ IDNO: 39) and EGFPRstoNB (CGGGATCCGCGGCCGCTTACTTGTACAGCTCGTCCATGCC/SEQ IDNO: 40). This was then inserted into the EcoRI-SacII site of theEcoRI-SacII fragment of the PCR product amplified by using pSA212 as atemplate, and the primers 5-2F (SEQ ID NO: 11) and 5-2R (SEQ ID NO: 12).

[EXAMPLE 4] Preparation of SIV on a Large Scale

[0094] Transfection

[0095] Cells from the cell line 293T, derived from human fetal kidneycells, (Proc. Natl. Acad. Sci. USA, vol. 90, pp8392-8396, 1993) wereplated in fifty 15-cm dishes at a cell density of 2.5×10⁶ cells/dish,and cultured for 48 hours in DMEM (GibcoBRL) containing 10% inactivatedfetal calf serum (FCS). Twenty ml of medium was used per 15-cm dish.After the cells had been cultured for two days, 300 μg. of the genetransfer vector pGCL3C/CMVL.U3G2/RREc/s/CMVEGFP/3′LTR,,U3, 150 μg of thepackaging vector pCAGGS/SIVagm gag-tat/rev, and 50 μg of the VSV-G(pVSV-G) expression vector were dissolved in 75 ml of OPTI-MEM(Invitrogen) Then 2 ml of PLUS reagent (Invitrogen) was added to thesolution. After stirring, the solution was allowed to stand at roomtemperature for 15 minutes. Three ml of LIPOFECTAMINE (Invitrogen) wasseparately mixed with 75 ml of OPTI-MEM, and then combined with the DNAmixture described above. The resulting mixture was allowed to stand atroom temperature for 15 minutes.

[0096] A 3-ml aliquot of this solution was added dropwise to each 293Tcell culture in which the medium had been replaced with 10 ml ofOPTI-MEM. The cells were incubated under 10% CO₂ at 37° C. for threehours. Ten ml of DMEM containing 20% FCS was added to each dish, and thecells were cultured for a further 21 hours. 24 hours after transfection,the medium of each dish was replaced with 20 ml of DMEM containing 10%FCS. The cells were cultured for a further 24 hours.

[0097] Recovery and Concentration of Vectors

[0098] The culture supernatant was saved and filtered with a 0.45-μmfilter, followed by centrifugation at 42500 g and 4° C. for 90 minutes.The resulting pellet was dissolved in 10 ml of TBS containing 10 mMMgCl₂, 3 mM spermine, 0.3 mM spermidine, and 100 μM dNTP, and then thesolution was incubated at 37° C. for two hours. The sample was thencentrifuged at 42500 g and 4° C. for two hours. The resulting pellet wassuspended in 1 ml of PBS containing 5% FCS and 2 μg/ml polybrene, andthen frozen and stored at −80° C.

[EXAMPLE 5] Preparation of Simian Blastocysts

[0099] In order to obtain blastocysts suitable for establishing EScells, fertilization was achieved using in-vitro fertilization and spermmicroinjection. The fertilized eggs were then manipulated for blastocystdevelopment using the in-vitro culture method.

[0100] (1) Ovary Stimulation Method

[0101] 1.8 mg of gonadotropin-releasing hormone (GnRH) [(Trade name:Leuplin (Takeda Chemical Industries, Ltd.; or Trade name: Sprecur(Hoechst Marion Roussel)] was given subcutaneously to female cynomolgusmonkeys (four to 15 years old). Two weeks after GnRH administration, thehormones indicated below were administered intramuscularly once a day atregular times (in the evening in this Example) for nine consecutivedays. These hormones were: pregnant mare serum gonadotropin (PMSG)[Trade name: Serotropin (Teikoku Hormone Mfg. Co., Ltd.)] at a dose of25 IU/kg; and human menopausal gonadotropin (hMG) [Pergonal (TeikokuHormone Mfg. Co., Ltd.) at a dose of 10 IU/kg; or follicle stimulatinghormone (FSH) [Fertinorm (Serono Laboratories)] at a dose of 3 IU/kg.After five days of administration, the growth of ovarian follicles wasconfirmed by observation of ovaries using a laparoscope (externaldiameter=3 mm).

[0102] After the monkeys had been administered with PMSG, hMG, or FSHand the ovarian follicles had been confirmed to have grown sufficiently,human chorionic gonadotropin (hCG) [Trade name: Puberogen (Sankyo CO.,Ltd.)] was administered intramuscularly a single time and at a dose of400 IU/kg. Eggs were collected 40 hours after hCG administration.

[0103] Egg collection was carried out by aspirating eggs together withfollicular fluid by puncturing the ovarian follicles using a 2.5-mlsyringe with a 60-mm 19G or 20G Cathelin puncture needle, whichcontained about 0.5 ml of an α-MEM (α-Modification of Eagle's Medium;ICD Biomedical Inc.) solution containing 10% SSS (Serum SubstituteSupplement; Irvine Scientific Sales Inc.). Egg collection was carriedout while observing the ovary with a laparoscope (external diameter=10mm).

[0104] Immediately after collection, mature eggs wrapped with cumuluscells were isolated under a stereoscopic microscope, and transferredinto TALP containing 0.3% BSA (hereinafter abbreviated as BSA/TALP) Theeggs were pre-cultured under 5% CO₂, 5% O₂, and 90% N₂ in a CO₂incubator at 37° C. for three to four hours.

[0105] (2) Sperm Collection

[0106] (i) Method of Collection from the Epididymis

[0107] The epididymis was collected from male cynomolgus monkeys (ten to15 years old) and a 1-ml syringe with a 23G needle was immediatelyinserted into the seminal duct. BWW containing 0.3% BSA (hereinafterabbreviated as BSA/BWW) was gently injected to the duct. The tail ofepididymis was cut and the seminal fluid which flowed from the duct wascollected.

[0108] (ii) Collection Method Using Electric Stimulation

[0109] (a) Rectal Method

[0110] Male cynomolgus monkeys (ten to 15 years old) were anesthetizedusing ketamine hydrochloride and xylazine hydrochloride (at doses of 5mg/kg and 1 mg/kg respectively), and allowed to lie in a supineposition. Keratin cream was applied to a rectal bar electrode connectedwith an electric stimulator, and the electrode was gently inserted intothe monkey's rectum. The penis was washed with sterilized physiologicalsaline, and dried with a paper towel or the like. The tip of the peniswas inserted into a test tube (50 ml). Then, five volts of AC electriccurrent was introduced using the electric stimulator. The cycle of poweron (for three to five seconds) and off (for five seconds) was repeatedup to three times. Voltage application was terminated when ejaculationoccurred during the cycle. When no ejaculation occurred, the sameprocedure was carried out using ten volts instead of five volts. Ifejaculation still had not occurred, the same procedure was repeated at15 volts, and likewise at 20 volts.

[0111] (b) Penis Method

[0112] Without anesthesia, the limbs of male cynomolgus monkeys (ten to15 years old) were held such that the monkeys were fixed to the front ofthe cage and the penis could be conveniently reached. With surgicallatex gloves, the penis was washed with sterile physiological saline andthen dried with paper towel or the like. An electric stimulator wasprepared, and electrodes were attached to the penis using clips. First,five volts of DC current was introduced at one-second intervals, andthen the intervals were gradually shortened. When no ejaculationoccurred, the same procedure was repeated at ten volts, and likewise atfifteen volts, and then at 20 volts. If ejaculation had still notoccurred, the same procedure was repeated using AC voltage.

[0113] (3) Method of post-treatment and cryopreservation of seminalfluid after collection (Torii, R., Hosoi, Y., Iritani, A., Masuda, Y.and Nigi, H. (1998). Establishment of Routine Cryopreservation ofSpermatozoa in the Japanese Monkey (Macaca fuscata), Jpn. J. Fertil.,43(2), 125-131).

[0114] Seminal fluid collected by the rectal method or the penis methodwas allowed to stand in a CO₂ incubator at 37° C. for about 30 minutes.The liquid components were saved, and about 1 to 2 ml of BWW culturemedium (Biggers, Whitten and Wittinghams) containing 0.3% BSA (BSA/BWW)was added to this liquid to prepare a sperm solution. This solution wasthen gently overlaid onto 2.5 ml of 80% Percoll (American PharmaciaBiotech Inc.) , and 2.5 ml of 60% Percoll. The resulting sample wascentrifuged at 1,400 rpm at room temperature for 20 minutes, and thenthe upper layer was removed by aspiration, leaving only about 0.5 ml atthe bottom of the test tube. About 10 ml of BSA/BWW was added to theliquid and the resulting mixture was gently mixed. After the mixture wascentrifuged at 1,400 rpm at room temperature for three minutes, theupper layer was removed by aspiration, leaving only about 0.5 ml at thebottom.

[0115] An appropriate amount of BSA/BWW was added to the collected spermto adjust the sperm density to about 5×10⁷ to 1.0×10⁸ cells/ml. Theresulting sperm solution was allowed to stand at 4° C. for about 60 to90 minutes. Then, a TTE-G solution [TTE medium (composition of the100-ml medium: 1.2 g of Tes, 0.2 g of Tris-HCl, 2 g of glucose, 2 g oflactose, 0.2 g of raffinose, 20 ml of egg yolk, 10,000 IU ofpenicillin-G, 5 mg of streptomycin sulfate) containing glycerol at thefinal concentration of 12%] having a ⅕^(th) of the volume of the spermsolution, was gently dropped into the sperm solution in iced water. Theresulting mixture was allowed to stand for five minutes, and then theabove-described cycle of dropping the TTE-G solution and standing wasrepeated five times.

[0116] After standing the mixture in iced water for 60 to 90 minutes,the resulting sperm solution was added to a 0.25- or 0.5-ml straw. Thestraw was held in the upper part of a liquid nitrogen container forabout five minutes, and then above the liquid nitrogen surface for fiveminutes. The straw was then stored in liquid nitrogen.

[0117] (4) Preparation of Sperm for In-Vitro Fertilization

[0118] Straws removed from liquid nitrogen were incubated at roomtemperature for 30 seconds, and then incubated in a 37° C. water bathfor 30 seconds to thaw the stored sperm solution. Then, 10 ml of BSA/BWWcontaining 1 mM caffeine (Sigma) and 1 mM dbC-AMP (Sigma) was added tothe straw, and the mixture was incubated in a CO₂ incubator (5% CO₂) at37° C. for 30 minutes, facilitating sperm capacitation.

[0119] The sperm liquid was centrifuged at 1,000 rpm (200×g) for twominutes, and the resulting supernatant was discarded. About 0.5 to 1 mlof BSA/BWW containing 1 mM caffeine and 1 mM dbC-AMP was added to thesperm. The sperm solution was allowed to stand in a CO₂ incubator (5%CO₂) at 37° C. for 60 minutes. The sperm which swam to the top werecollected, and sperm count and motility were examined. Thus, in this waysperm for in-vitro fertilization were prepared.

[0120] (5) Fertilization Method

[0121] (A) In-Vitro Fertilization Method

[0122] One to five eggs wrapped with cumulus cells were transferred into50-μl BSA/BWW spots, which were covered with mineral oil and in aplastic dish. Then, the sperm suspension was transferred into each dropat a density of 5.0×10⁵ to 1.0×10⁶ cells (sperms) /ml. The drops werecovered with mineral oil, after which insemination took place.

[0123] After fertilization, the eggs were cultured in a CO₂ incubatorwith 5% CO₂, 5% O₂, and 90% N₂ at 37° C. Five hours after insemination,TALP solution was substituted for the BWW solution. Fertilizationefficiency was determined to be about 45%, and thus fertilized eggs wereyielded with a high efficiency. Eggs in which fertilization wasconfirmed were cultured for about 20 hours. The eggs were thentransferred into a CMRL-1066 solution and further cultured.

[0124] The CMRL-1066 solution was prepared as follows: 0.014615 g ofL-glutamine (1 mM) was dissolved in 10 ml of solution A [penicillin G(1000 units), 0.5 ml of gentamicin sulfate (10 mg/ml), 10 ml ofCMRL-1066 (10×) (without NaHCO₃ and L-glutamine), 0.218 g of NaHCO₃; 6.7ml of sodium lactate (290 mOsmol's stock); adjusted to 100 ml withwater]. The solution thus prepared was sterilized by filtration.Solution B (10 ml) was prepared by adding 9 ml of solution A to 1 ml ofthe sterilized solution. Solution C was prepared by dissolving 0.0055 gof sodium pyruvate (final concentration=5 mM) in solution B. 8 ml ofsolution C was combined with 2 ml of BCS (bovine calf serum). Theresulting mixture was sterilized by filtration to prepare CMRL-1066solution.

[0125] (B) Sperm Microinjection

[0126] (i) Egg Preparation

[0127] The harvested eggs were placed in a 50-μl spot of TALP (BSA/TALP)solution containing 0.3% BSA covered with mineral oil (Sigma), andpre-cultured in 5% CO₂, 5% O₂, and 90% N₂ at 37° C. for about two tofour hours.

[0128] To confirm egg maturity, the egg culture was incubated for oneminute in a TALP-HEPES solution containing 0.1% hyaluronidase (Sigma)Cumulus cells were then removed by pipette. The recovered eggs werecategorized under an inverted microscope into the following four classes(Class-1 to -4):

[0129] Class-1: mature eggs having polar bodies (PB)

[0130] Class-2: eggs in the middle of the maturation process, without PBor germinal vesicles (GV)

[0131] Class-3: premature eggs containing GV

[0132] Class-4: eggs markedly distorted or with degenerated cytoplasmcomprising retrogressive changes

[0133] Immediately after categorization, Class-1 eggs were used inmicroscopic fertilization. Class-2 and Class-3 eggs were placed in 50-μlspots of BSA/TALP solution covered with mineral oil, and then furthercultured in 5% CO₂, 5% O₂, and 90% N₂ at 37° C. 24 hours after culture,egg maturation was confirmed. Matured eggs were used in microscopicfertilization at this time. The remaining premature eggs and Class-4eggs were not used in fertilization.

[0134] (ii) Sperm Preparation

[0135] Sperm were prepared according to the method described in the“In-vitro fertilization method” section.

[0136] (iii) Sperm Microinjection

[0137] Microscopic fertilization was carried out under an invertedmicroscope (Olympus IX70) equipped with a micro-manipulator fromNarishige.

[0138] In a 15-cm dish, spot 1 (15 μl of a diluted sperm solution), spot2 (3×5 μl of 10% polyvinylpyrrolidone/PBS culture medium [PVP; meanmolecular weight=about 360,000 (Nacalai Tesque)], and spot 3 (3×5 μl ofTALP-HEPES (BSA at a final concentration of 3 mg/ml) solution for eggmanipulation) were placed successively, and the spot surfaces werecovered with mineral oil to prevent drying. Microscopic fertilizationwas performed using this dish. No heating devices were used in thisExample, and any temperature changes during manipulation were ignored.However, it is possible to use a heating device.

[0139] The injection needle used was that used for microscopicfertilization in humans, and was set to an inclination angle of 30degrees (external diameter, 7 to 8 μm; internal diameter, 5 to 7 μm(Medi-Con International Co., Ltd. . The above-mentioned needle wasconnected with a high precision Alcatel syringe.

[0140] The needle used to hold the egg was the same as that used formicroscopic fertilization in humans, and was set to an inclination angleof 30 degrees. Alternatively, a needle (external diameter=about 100 μm;internal diameter at the end=about 15 μm) prepared using a magneticpuller (Trade name: PN-30, Narishige) was used. The above-mentionedneedles were connected with Narishige injectors that had a 2000-μlair-tight syringe.

[0141] Sperm with a high motility were selected according to the samecriteria used for microscopic fertilization in humans, and thenaspirated from spot 1. The selected sperm were transferred into spot 2.In spot 2, sperm motility was reduced due to PVP viscosity. To preventthe sperm from moving, the sperm membranes were partially disrupted byrubbing the sperm tails with the injection needle. The sperm wereaspirated together with the viscous solution, and then transferred intospot 3.

[0142] Matured eggs were placed in spot 3, and then fixed at the 6 or 12o'clock position using the holding needle, so as not to disrupt thechromosomes under the polar bodies with the injection needle. Then, asperm was placed at the tip of the injection needle, and inserted intothe egg. After confirming that the needle had passed through the zonapellucida, the egg cell membrane was aspirated. Membrane rupture wasconfirmed, and then the contents of the injection needle (the sperm andegg cytoplasm) were injected into the egg. This procedure of injectingsperm and egg cytoplasm was carried out repeatedly. Two to three eggswere fertilized in a single manipulation. However, if the inner surfaceof the needle tip became clogged with sperm or egg cytoplasm, the tipwas washed with the liquid in spot 2.

[0143] The microscopically fertilized eggs were immediately placed in anincubator and cultured in 5% O₂, 5% CO₂, and 90% N₂ at 37° C.Immediately after microscopic fertilization, 50-μl spots of CMRL-1066solution were created in an uncoated 6-cm culture dish and covered withparaffin oil. The spots were typically equilibrated with a gas. phasefor at least three hours. 24 hours after microscopic fertilization, theeggs were transferred from the TALP solution into a spot of theabove-mentioned CMRL-1066 solution, and then incubated in 5% O₂, 5% CO₂,and 90% N₂ in a tightly sealed CO₂ incubator at 37° C. for eight days.Fertilized eggs were produced with high fertilization efficiency (about75 to 85%).

[0144] (6) Culture Method

[0145] Following in-vitro fertilization and microscopic fertilization,eggs confirmed to be fertilized were cultured using hanging microdropcultures in which the culture medium was covered with mineral oil toavoid abrupt changes in temperature and carbon dioxide concentration.This method has been widely used for experimental animals such as miceand rabbits, but has not been routinely used for humans. The eggs werecultured under air tight conditions to avoid unnecessary stresses causedby temperature and pH changes. Thus, opening and closing of theincubator door was avoided until blastocyst formation was predicted,namely for seven days after the start of the in-vitro fertilizationcultures, or for eight days after the start of the microscopicfertilization cultures.

[0146] The medium, temperature, and gas phase used for the cultures wereas follows:

[0147] Culture Medium: TALP & CMRL-1066

[0148] The mediums used were BWW, which is routinely used for mice, andP1 (Nakamedical Inc.) , Blast medium (Nakamedical Inc.) , and the newlydeveloped HFF (human foilcular fluid; Fuso Pharmaceutical Industries,Ltd.), which are used for humans. When cultured in these mediums, eggfertilization and segmentation progressed normally until the developmentstopped at the morula stage. After confirmation of fertilization, acombination of TALP and CMRL-1066 culture media was applied, andfertilized embryos went on to form blastocysts at an exceedingly highrate of 40 to 46%. The use of the HEPES buffer system TALP instead ofPBS, a phosphoric acid buffer system, in manipulations outside theincubator was presumed to reduce adverse effects on the eggs.

[0149] Culture Temperature: 38° C.

[0150] Mouse and human embryos are routinely cultured at 37° C. However,at this temperature, the eggs only developed slowly and the developmentstopped at the morula stage. The eggs were then cultured at the slightlyhigher temperature of 38° C., which is similar to the temperature of38.5° C. used for culturing bovine embryos, etc. Blastocysts wereproduced seven days after in-vitro fertilization, and eight days aftermicroscopic fertilization.

[0151] Culture Atmosphere: 5% CO₂/5% O₂/90% N₂

[0152] Under the typical conditions of 5% CO₂ and 95% air, the eggsstopped developing farther than the morula stage. However, when culturedin 5% CO₂, 5% O₂, and 90% N₂, the eggs were revealed to form blastocystswith a high efficiency.

[0153] The TALP solution and TALP-HEPES solution were prepared asfollows: TABLE 1 STOCK SOLUTION(ml) STOCK SOLUTION FINAL SOLUTION TALP-REAGENT (mM) (g/100 mol) (mM) TALP HEPES HEPES — 10.0 — 240 mg NaCl157.0 0.92 114.0 to 100 ml to 100 ml KCl 166.0 1.24 3.16 1.9 1.9 CaCl₂120.0 1.76 2.0 1.7 1.7 MgCl₂.6H₂O 120.0 2.44 0.5 0.41 0.41 SODIUMLACTATE 150.0 — 10.0 6.7 6.7 WATER — — — 7.1 NaH₂PO₄.H₂O 20.5 — 0.35 1.71.7 GLUCOSE 295.0 5.31 5.0 NaHCO₃ 167.0 1.40 25.0(TALP) 15.0 1.22.0(TALP-HEPES)

[0154] Just prior to preparing the TALP solution, the reagents listedbelow were prepared and sterilized with filters. Sodium pyruvate 0.5 mM0.0055 g (per 100 ml) Gentamicin sulfate (10 mg/ml) 50 μg/ml 50 μl BSA 3mg/ml 0.3 g

[0155] Just prior to preparing the TALP-HEPES solution, the reagentslisted below were prepared and sterilized with filters: Sodium pyruvate0.1 mM 0.0011 g (per 100 ml) BSA 3 mg/ml 0.3 g

[0156] When preparing the TALP-HEPES solution, 50 ml of NaCl andNa-HEPES (N-2-hydroxyethyl piperazine-N′-2-ethane sulfonate), phenolred, and penicillin G were first dissolved. The required aliquot of eachstock solution was added to the resulting solution, and finally thevolume was adjusted to 100 ml using NaCl stock solution. The pH of theresulting solution was adjusted to 7.4 using 1M NaOH. A solution ofsodium lactate was prepared by combining a stock solution (60% syrup)with water at the ratio of 1:35. After 1 mg/ml phenol red was added tothe resulting solution, the pH of the mixture was adjusted to 7.6 using1M NaOH, and then sterilized by filtration. The reagent thus preparedcan be stored at 4° C. for one week. 28 mg of NaHPO₄.H₂O was dissolvedin 10 ml of glucose solution, and the resulting solution was sterilizedby filtration. This solution can also be stored at 4° C. for one week.

[0157] The composition of BWW (Biggers, Whitten and Whittingham)solution is shown in Table 2. TABLE 2 REAGENT AMOUNT* (mg) SODIUMCHLORIDE 2,770 POTASSIUM CHLORIDE 178 KH₂PO₄ 81 MAGNESIUM SULFATE 147NaHCO₃ 1,053 SODIUM PYRUVATE 14 D(+)-GLUCOSE(ANHYDROUS) 500 PENICILLIN G31 STREPTOMYCIN 25 DL-SODIUM LACTATE 1,037 CALCIUM LACTATE 263 PHENOLRED 1 mg Merk 1

[EXAMPLE 6] Method for Establishing Simian ES Cells

[0158] (1) Preparation of Feeder Cells

[0159] Primary embryonic fibroblast cells (hereinafter also referred toas MEF cells) prepared from 12.5 day-old mouse embryos were culturedfrom the first to the third generation in MEM containing 10% fetalbovine serum (FBS), until the cells were confluent. To inactivate celldivision, MEF cells were then cultured for two or three hours in MEMcontaining mitomycin C (MMC) at a final concentration of 10 μg/ml. Theculture medium containing MMC was removed, and the cells were washedthree times with PBS. After washing, the cells were harvested from theculture dish by trypsinization (0.05% trypsin and 1 mM EDTA) and thecell count was determined.

[0160] MMC-treated MEF cells were plated at a cell density of 2×10⁴cells/well into each well of 24-well gelatin-coated culture dishes.

[0161] The cells obtained were plated on dishes to confirm that the cellcounts were sufficient, and then murine ES cells were cultured on theMEF cells, and cell properties were examined. The MEF cells wererevealed to be suitable as feeder cells because the cells had excellentgrowth capacity and still remained undifferentiated. Cells in the thirdgeneration or younger (the first to third generation) were suitable asfeeder cells.

[0162] (2) Separation of the Inner Cell Mass from Simian Blastocysts

[0163] To remove the zona pellucida, simian blastocysts were transferredinto M2 culture medium containing pronase or Tyrode at a finalconcentration of 0.5% [see; for example, D. M. Glover et al., Eds., DNACloning 4 Mammalian Systems A Practical Approach 2nd Ed. (1995)], andincubated at 37° C. for ten minutes. Blastocysts still covered with thezona pellucida were treated with pronase at 37° C. for five minutes.After confirming that the zona pellucida was removed, the blastocyststhus obtained were washed twice with PBS.

[0164] Then, a rabbit anti-cynomolgus monkey lymphocyte antiserum wasdiluted 20 times with M16 culture medium [see “DNA Cloning 4 MammalianSystems A Practical Approach” indicated above, and others]. Blastocystswere transferred into this diluted solution and incubated at 37° C. for30 minutes. The blastocysts thus obtained were washed three times withPBS. The blastocysts were transferred into a solution of complementdiluted 50 times with M16 culture medium and then incubated at 37° C.for 30 minutes. The blastocysts were then washed three times with PBS.When the trophectoderm was not completely removed from the blastocysts,it was removed mechanically using a glass needle under a microscope.Thus, the inner cell mass (ICM) was isolated by the procedure describedabove.

[0165] (3) Culture of Monkey Inner Cell Mass

[0166] MEM was removed from the 24-well culture dishes in which thefeeder cells prepared in section (1) had been plated. An 800-μl aliquotof ES cell culture medium [ES cell culture medium: Table 3] was added toeach well.

[0167] ICM prepared in section (2) was transferred into each well at acell density of one cell/well using a micropipette, and the cells wereincubated under 5% CO₂ at 37° C. for seven days. To avoid inhibition ofICM implantation, the medium was not changed for the first three daysafter the start of the culture, and the implantation was monitored everyday under a microscope. TABLE 3 COMPOSITION OF ES CELL CULTURE MEDIUMPRODUCT NAME AMOUNT DMEM/F12 (SIGMA) 500 ml FBS (JRH BIOSCIENCES) 75 mlGLUTAMINE (SIGMA; 200 mM) 5 ml PENICILLIN (SIGMA; 10.000 IU/ml) AND 5 mlSTREPTOMYCIN (SIGMA; 10 mg/ml) MIXTURE SODIUM PYRUVATE (SIGMA; 100 mM) 5ml SODIUM BICARBONATE (SIGMA; 7.5%) 8 ml 2-MERCAPTOETHANOL (SIGMA; FINALCONCENTRATION 10⁻⁴ M) 4 μl LIF(ESGRO; FINAL CONCENTRATION 0.5 ml (10⁶U/ml) 1000 U/ml)

[0168] On day 7 of the culture, the ICM had dispersed into single cells.The ES cell culture medium was removed from each well, and the wellswere washed once with PBS. 300 μl of a 0.25% trypsin/0.02% EDTA solutionwas added to each well, and then immediately removed. The 24-wellculture dishes were then incubated at 37° C. for one minute. After celldispersal was confirmed under a microscopes 500 μl of ES cell culturemedium was added to each well. The cells were dispersed by thoroughpipetting.

[0169] All of the cells described above were transferred into the wellsof fresh 24-well culture dishes in which feeder cells had been plated inadvance. 300 μl of ES cell culture medium was added to each well, andthus each well contained 800 μl of culture medium in total. Then, themedium was thoroughly mixed to plate the cells evenly. The ES cellculture medium was changed once every two days. Cell populationspresumed to comprise ES cells grew in less than seven days followingcell dispersion, and were observed every day for the appearance ofcolonies. When an ES cell colony appeared, the cells in that 24-wellculture plate were trypsinized, and then further subcultured. Duringthis period, the ES cell culture medium was changed every day or onceevery two days. As a result, a number of ES cell lines were yielded fromthe blastocysts of cynomolgus monkeys.

[0170] (4) Assessment of Simian ES Cells

[0171] Karyotype:

[0172] The number of chromosomes in the ES cells were examined todetermine if they were normal (the number of chromosomes was the same asfor the monkeys from which the ES cells were obtained: 2n=42). Theresults showed that the established ES cell strains were of normalkaryotype.

[0173] Pluripotency:

[0174] 1×10⁶ cynomolgus monkey ES cells were given to an 8-week old SCIDmouse by subcutaneous injection in the groin region. The formation of atumor was observed 5 to 12 weeks after the injection. The tumor wasfixed with Bouin's fixative or paraformaldehyde solution, and slicedinto sections. The sections were stained with hematoxylin and eosin (HEstain), or immuno-stained for histological examination. Since there werevery few monkey tissue-specific antibodies available for theimmuno-staining, antibodies against human neuron-specific enolase (NSE), glia fibrillary acidic protein (GFAP) , S-100 protein, and desmin wereused.

[0175] The tumors were revealed to be teratomas comprising cell typesderived from the ectoderm (neurons and glia) , mesoderm (muscle,cartilage, and bone), and endoderm (ciliated epithelium and intestinalepithelium). In the immunohistological examination, neurons weredetected using an antibody against NSE; glia were detected usingantibodies against NSE and GFAP; peripheral neurons were detected usingan antibody against NSE; cartilage was detected using an antibodyagainst S-100 protein; and muscle was detected using an antibody againstdesmin. The findings described above show that the cynomolgus monkey EScells have pluripotency (tridermic differentiation potency).

[0176] Morphological Features:

[0177] 1. The ES cells were characterized by a high nucleus/cytoplasmratio, notable nucleolar and colony formations. 2. The colony wasflatter in shape than the mouse ES cells.

[0178] Expression of Cell Surface Markers:

[0179] To test the presence of stage-specific embryonic antigens (SSEA)which are cells surface markers used to characterize ES cells, the EScells were immuno-stained using antibodies against the respective cellsurface markers for SSEA-1 (negative control) , SSEA-3, and SSEA-4.These antibodies are available from “The Developmental Studies HybridomaBank of the National Institute of Child Health and Human Development”.The ES cells were assessed for each SSEA cell surface marker using theprocedure described below: The cells were fixed with 4%paraformaldehydeand incubated with a primary antibody. The cells were then incubatedwith a labeled polymer (Simple Stain PO, Nichirei) comprising an aminoacid polymer conjugated with peroxidase and a secondary antibody.Detection was carried out by adding Simple Stain DAB solution(Nichirei). While SSEA-1 was undetectable, SSEA-3 and SSEA-4 weredetected.

[0180] Alkaline Phosphatase Activity:

[0181] Alkaline phosphatase activity was assayed using HNPP (Roche) andFast-Red TR SaH as a substrate. Alkaline phosphatase was detected.

[EXAMPLE 7] Culture of Simian ES Cells

[0182] (A) Preparation of Feeder Cells

[0183] In the same way as described above in Example 6, MEF cells wereobtained from 12.5 day-old mouse embryos and cultured in MEM containing10% FBS and from the first to the third generation until confluent. Toinactivate cell division, MEF cells were cultured for two or three hoursin MEM containing MMC at a final concentration of 10 μg/ml. Then, theculture medium containing MMC was removed and the cells were washedthree times with PBS. The cells were harvested by trypsinization (0.05%trypsin/1 mM EDTA) , and the cell count was determined.

[0184] MMC-treated 2×10⁴ MEF cells were plated in each well of 24-wellgelatin-coated culture dishes.

[0185] (B) Culture of Simian ES Cells (CMK-1 Strain)

[0186] An ES cell culture medium was prepared according to the proceduredescribed in Example 6. CMK-1 strain ES cells (hereinafter also referredto as “CMK-1”) were plated on feeder cells prepared by the proceduredescribed above in section (A). At this point, the ES cells were notdispersed into single cells but plated as cell masses of 5 to 10 cells.The culture medium was changed daily or every two days. The cells werepassaged every 4 to 6 days.

[0187] On passaging, the cells were washed once with PBS, and 0.25%trypsin/PBS or 0.1% collagenase/DMEM was added to the cells, followed byincubation 37° C. for two to ten minutes. The cells were suspended in EScell culture medium, and centrifuged at 1000 rpm for five minutes. Thecells were then plated on freshly prepared feeder cells at a cell ratioof 1:2 to 1:4. A commercially available solution for cellcryopreservation, or 10 to 20% DMSO/DMEM was used to store the cells.

[EXAMPLE 8] <Gene Transfer Experiments Using Simian ES Cells (CMK-1Strain)>

[0188] <Gene Transfer into Simian ES Cells (CMK-1 Strain)>

[0189] A gene was introduced into simian ES cells using theVSV-G-pseudotyped SIV vector [self-inactivating (SIN) vector] for EGFPexpression (FIG. 4) prepared by the procedure described above. Thefunctional titer of the vector solution was 1.9×10⁹/ml.

[0190] On the day before gene transfer, at a cell density of 7.5×10⁴cells/ml, CMK-1 cells were plated on to feeder cells (2.5×10⁵ cells/ml)On the day of gene transfer (Day 0) , the SIV vector described above wasdiluted with the ES cell culture medium (Example 6) to adjust the MOI to1, 10, and 100, based on the cells prepared as described above.Polybrene was added at a concentration of 8 μg/ml, and transduction wascarried out once. The medium was changed after ten hours. From the nextday (Day 1) , the cells were passaged on to feeder cells every five tosix days at a cell ratio of 1:3 to 1:4.

[0191] The efficiency of gene transfer into ES cells (CMK-1 strain) wasdetermined according to the formula below, which is based on the EGFPexpression level as estimated by FACScan. Since the CMK-1 cells werecultured on feeder cells, samples recovered using trypsin contained bothCMK-1 and feeder cells. In addition, the SIV vector can deliver genesinto mitomycin C-treated feeder cells as well as into CMK-1 cells.Therefore cells expressing EGFP included both CMK-1 and feeder cells.EGFP expression level was determined only in CMK-1 cells and accordingto the procedures described below in sections “(A) Assay for theexpression level of EGFP in feeder cells” and “(B) Determination of cellratio of CMK-1 and feeder cells” using the normalization equationdescribed below.

[0192] <Normalization Equation for the Efficiency of Gene Transfer forCMK-1>

[0193] In each FACS sample, EGFP expression level in CMK-1 cells isrepresented by Ec; EGFP expression level in feeder cells is representedby Ef; and EGFP expression level in the mixture of CMK-1 and feedercells is represented by Eb. If c denotes the number of CMK-1 cells and fdenotes the number of feeder cells:

f·Ef+c·Ec=(f+c)Eb

[0194] A rearrangement of the formula gives the following equation:

Ec={(f+c)Eb−f·Ef}/c

[0195] Assuming c/(f+c)=k yields:

1/k=1+f/c

[0196] Substituting this equation in the above equation yields thefollowing: EC = Eb/k − (1/k − 1)/Ef   = (Eb − Ef)/k + Ef

[0197] This gives the equation shown below:

mean Ec={(Eb−Ef)/n·,,1/k _(i) }+Ef

[0198] $\begin{matrix}{{{Standard}\quad {error}\quad {SEM}}\quad} \\\left( {{srandard}\quad {error}\quad {of}\quad {mens}} \right)\end{matrix} = {{{1/n} \cdot \left( {{}_{}^{}\left( {{Ec}_{i} - {mean}} \right)_{}^{}} \right)^{1/2}}\quad = {\left( {{Eb} - {Ef}} \right) \cdot \left( {{}_{}^{}\left( {{1/k_{i}} - {{1/n}{{\,_{''}1}/k_{i}}}} \right)_{}^{}} \right)^{1/2}}}$

[0199] Thus, the normalization equation described above is reached.

[0200] (A) Assay for EGFP Expression in Feeder Cells

[0201] The SIV vector was only introduced into feeder cells, and atMOI=1, 10, and 100. The cells were passaged and FACS analysis wascarried out by the same procedures as described above.

[0202] (B) Determination of the Cell Ratio for CMK-1 and Feeder Cells

[0203] CMK-1 cells were distinguished from feeder cells using the methoddescribed below. The anti-HLA-ABC antibody, which is an antibody againsthuman HLA (mouse anti Human HLA-ABC: RPE, Serotec Ltd.), does not reactto mouse-derived feeder cells, but does react to cynomolgusmonkey-derived CMK-1 cells. This antibody was allowed to react with asuspension of CMK-1 and feeder cells, and the ratio between CMK-1 andfeeder cells was determined by FACS.

[0204] The efficiency of SIV vector-mediated gene transfer into simianES cells was determined (FIG. 5). The efficiency of gene transfer intoCMK-1 cells was corrected to eliminate the contribution of feeder cellcontamination, using the method described above in the section“normalization equation for the efficiency of gene transfer for CMK-1”.The efficiency of gene transfer was dependent on MOI and was exceedinglyhigh two days after gene transfer. At MOI=100 the efficiency was 90% orhigher; at MOI=10 it was about 80%; and at MOI=1 it was about 60%. Thishigh transgenic efficiency lasted for at least about two months. A timecourse of mean fluorescence intensity was determined in CMK-1 cells intowhich the EGFP gene had been introduced. The mean fluorescence intensityof EGFP-expressing cells was hardly reduced over about two months (FIG.6). Micrographs for the CMK-1 cells (“green” ES cells) in which the EGFPgene had been introduced via the SIV vector are shown in FIG. 7.

[EXAMPLE 9] Efficiency of SIV Vector-Mediated Gene Transfer into SimianES Cells and Murine ES Cells

[0205] To test whether the efficiency of SIV vector-mediated genetransfer depends on the species from which the ES cells were derived,genes were introduced into murine ES cells (D3 strain) by the samemethod as described in Example 8.

[0206] On the day before gene transfer, CMK-1 cells were plated at acell density of 7.5×10⁴ cells/ml on to feeder cells (2.5×10⁵ cells/ml)On the day of gene transfer (Day 0), the above-describedVSV-G-pseudotyped SIV vector [self-inactivating (SIN) vector] for EGFPexpression (FIG. 4) was diluted with the ES cell culture medium (Example6) to adjust the MOI to 1, 10, and 100, based on the cells prepared asdescribed above. Polybrene was added at a concentration of 8 μg/ml, andtransduction was carried out once. The medium was changed after tenhours. From the next day (Day 1), the cells were passaged. on to feedercells every five to six days at a cell ratio of 1:3 to 1:4. On the daybefore gene transfer, murine ES cells (D3 strain) were plated at a celldensity of 1×10⁵ cells/ml and on nearly the same number of feeder cells.The same type of feeder cell as for CMK-1 was used. The day after theplating, the above-described SIV vector was added to the medium atMOI=10. Polybrene was added at a concentration of 8 μg/ml, andtransduction was carried out once. From the next day (Day 1) , the EScells were passaged on feeder cells every other day. The cell ratio was1:8 to 1:10.

[0207] The efficiency of gene transfer into each ES cell type at MOI=10is shown in FIG. 8. The efficiency of gene transfer into simian ES cellswas found to be higher than that into murine ES cells (FIG. 8). When anSIV-based vector is used, genes are thought to be introduced moreefficiently into primate cells such as SIV's natural host monkey cells,than into cells from a different species such as mouse.

[0208] Industrial Applicability

[0209] The VSV-G-pseudotyped simian immunodeficiency virus vector forgene transfer into primate ES cells of the present invention is usefulfor research into embryology and disease, clinical applications, andexperimental models for primates including humans. Furthermore, thevector of the present invention enables screening for genes, reagents,and such, which control the specific differentiation of tissues or cellsfrom ES cells. This screening method is highly advantageous in selectinggenes, reagents, and the like, which are involved in the specificdifferentiation of tissues or cells, and which are useful in preparingdesired differentiated cells or tissues.

1 40 1 36 DNA Artificial Sequence Description of Artificial Sequenceartificially Synthesized Primer Sequence 1 gcagatctca accaggaggcgaggctgcat tttggg 36 2 36 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 2gcgaattcta cttactggtg ctgtaaagga gccaaa 36 3 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 3 atcggaattc ttttattgta agatggattg gtttttaaat 40 4 48 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 4 cgggatccgc ggccgcggat atggatctgtggagatagag gaacatat 48 5 29 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 5tcgagactag tgacttggtg agtaggctt 29 6 29 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 6 tcgaaagcct actcaccaag tcactactc 29 7 19 DNA ArtificialSequence Description of Artificial Sequence artificially SynthesizedOligonucleotide Sequence 7 aatttctcga gcggccgca 19 8 19 DNA ArtificialSequence Description of Artificial Sequence artificially SynthesizedOligonucleotide Sequence 8 aatttgcggc cgctcgaga 19 9 35 DNA ArtificialSequence Description of Artificial Sequence artificially SynthesizedPrimer Sequence 9 gcggtacctg gatgggattt attactccga tagga 35 10 40 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 10 gcgaattcga tagggcttga aacatgggtactatttctgc 40 11 36 DNA Artificial Sequence Description of ArtificialSequence artificially Synthesized Primer Sequence 11 gcgaattcccgtttgtgcta gggttcttag gcttct 36 12 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 12 tccccgcgga tatggatctg tggagataga ggaacatatc 40 13 44 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 13 gcgcggccgc ggatccgtcg acgcactttttaaaagaaaa ggga 44 14 36 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 14gcgagctcta atgcaggcaa gtttattagc tttcta 36 15 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 15 ggaattcccg cggtagttat taatagtaat caattacggg 40 16 40 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 16 cgggatccgc ggccgcttac ttgtacagctcgtccatgcc 40 17 40 DNA Artificial Sequence Description of ArtificialSequence artificially Synthesized Primer Sequence 17 atcggaattcttttattgta agatggattg gtttttaaat 40 18 50 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 18 ataagaatgc ggccgctagc taagctgaat gaggagggtc aggcaactgt 50 1936 DNA Artificial Sequence Description of Artificial Sequenceartificially Synthesized Primer Sequence 19 gcgaattccc gtttgtgctagggttcttag gcttct 36 20 48 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 20agctagctag gctagcggat atggatctgt ggagatagag gaacatat 48 21 39 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 21 tatataagca gagctcgctg gcttgtaactcagtctctt 39 22 40 DNA Artificial Sequence Description of ArtificialSequence artificially Synthesized Primer Sequence 22 tatataagtgcagtacgctg gcttgtaact cagtctctta 40 23 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 23 tataaaaagc gaagccgctg gcttgtaact cagtctctta 40 24 40 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 24 gcgaattcga tagggcttga aacatgggtactatttctgc 40 25 40 DNA Artificial Sequence Description of ArtificialSequence artificially Synthesized Primer Sequence 25 cggggtacctcaatattggc cattagccat attattcatt 40 26 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 26 agttacaagc cagcgagctc tgcttatata gacctcccac 40 27 35 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 27 gcggtaccta gttattaata gtaatcaatt acggg 3528 40 DNA Artificial Sequence Description of Artificial Sequenceartificially Synthesized Primer Sequence 28 agttacaagc cagcgagctctgcttatata gacctcccac 40 29 35 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 29gcggtaccag gctccccagc aggcagaagt atgca 35 30 40 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 30 agttacaagc cagcgtactg cacttatata cggttctccc 40 31 40 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 31 ggggtaccat tgattattga ctagttattaatagtaatca 40 32 40 DNA Artificial Sequence Description of ArtificialSequence artificially Synthesized Primer Sequence 32 agttacaagccagcggcttc gctttttata gggccgccgc 40 33 99 DNA Artificial SequenceDescription of Artificial Sequence artificially Synthesized PrimerSequence 33 atgcgagctc gtcgacgcac tttttaaaag aaaagggagg actggatgggatttattact 60 ccgataggac gctggcttgt aactcagtct cttactagg 99 34 36 DNAArtificial Sequence Description of Artificial Sequence artificiallySynthesized Primer Sequence 34 gcgagctcta atgcaggcaa gtttattagc tttcta36 35 99 DNA Artificial Sequence Description of Artificial Sequenceartificially Synthesized Primer Sequence 35 atgcgagctc gtcgacgcactttttaaaag aaaagggagg actggatggg atttattact 60 ccgataggat caatattggccattagccat attattcat 99 36 99 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 36atgcgagctc gtcgacgcac tttttaaaag aaaagggagg actggatggg atttattact 60ccgataggaa ggctccccag caggcagaag tatgcaaag 99 37 99 DNA ArtificialSequence Description of Artificial Sequence artificially SynthesizedPrimer Sequence 37 atgcgagctc gtcgacgcac tttttaaaag aaaagggaggactggatggg atttattact 60 ccgataggac attgattatt gactagttat taatagtaa 9938 36 DNA Artificial Sequence Description of Artificial Sequenceartificially Synthesized Primer Sequence 38 gcgagctcta atgcaggcaagtttattagc tttcta 36 39 40 DNA Artificial Sequence Description ofArtificial Sequence artificially Synthesized Primer Sequence 39atcggaattc ggccgccatg gtgagcaagg gcgaggagct 40 40 40 DNA ArtificialSequence Description of Artificial Sequence artificially SynthesizedPrimer Sequence 40 cgggatccgc ggccgcttac ttgtacagct cgtccatgcc 40

1. A recombinant simian immunodeficiency virus vector pseudotyped withVSV-G and able to introduce a gene into a primate embryonic stem cell.2. The vector according to claim 1, wherein the recombinant simianimmunodeficiency virus vector is derived from the agm strain.
 3. Thevector according to claim 1 or 2, wherein the recombinant simianimmunodeficiency virus vector is a self-inactivating vector.
 4. Thevector according to any one of claims 1 to 3, wherein the primatebelongs to the Old World primates, of the family Cercopithecidae, genusMacaca.
 5. The vector according to any one of claims 1 to 4, whichcarries a foreign gene in an expressible state.
 6. The vector accordingto claim 5, wherein the foreign gene is a gene encoding a proteinselected from the group consisting of green fluorescent protein,β-galactosidase, and luciferase.
 7. A method for introducing a gene intoa primate embryonic stem cell, which comprises the step of contactingthe cell with the recombinant simian immunodeficiency virus vectoraccording to any one of claims 1 to
 6. 8. A primate embryonic stem cellinto which the recombinant simian immunodeficiency virus vector,according to any one of claims 1 to 6, has been introduced.
 9. A cellyielded by allowing the primate embryonic stem cell according to claim 8to proliferate and/or differentiate.
 10. A method for detecting theeffect of an introduced gene on the proliferation or differentiation ofES cells, which comprises the steps of: (a) introducing the vector,according to any one of claims 1 to 6, into a primate embryonic stemcell; and (b) detecting the proliferation or differentiation of theembryonic stem cell.